Evaluation of microbiological and physico–chemical quality of water from aquifers in the North West Province, South Africa

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Evaluation of microbiological and physico-chemical quality of

water from aquifers in the North West Province, South Africa

By

Alewyn Johannes Carstens 20143508

Submitted in fulfilment of the requirement for the degree of MAGISTER OF SCIENCE IN ENVIRONMENTAL SCIENCE

(M.Sc Env.Sci)

Faculty of Science

North West University, Potchefstroom Campus

Potchefstroom, South Africa

Supervisor: Prof. C.C. Bezuidenhout Potchefstroom

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ABSTRACT

Contamination of groundwater that is suitable for drinking is of growing concern as the water supply of South Africa is becoming increasingly limited. This is especially the case in the North West province, with its semi – arid climate and variable rainfall patterns. The aim of the study was to evaluate the microbiological and physico – chemical qualities of groundwater obtained from selected DWA (Department of Water Affairs) monitoring boreholes in the Mooi River and Harts River catchment areas. Physico-chemical parameters included temperature, pH, electrical conductivity (EC), salinity, total dissolved solids (TDS), sulphate and nitrate concentrations. Physical parameters were measured using a calibrated submerge-able multimeter and chemical parameters using specialised kits and a spectrophotometer. Microbiological parameters included heterotrophic plate counts and total and faecal coliform enumeration. Membrane filtration and culture based methods were followed for enumeration of bacteria. During the identification procedures multiplex PCR for E. coli identification and 16S rRNA gene sequencing for identification of heterotrophic plate count bacteria and amoeba resistant bacteria were used. For antibiotic resistance, the Kirby-Bauer (1996) disk diffusion method was used. During the warm and wet season high electrical conductivity and salinity were observed in the Trimpark (65.3 mS/m; 325 ppm), School (125.1 mS/m; 644 ppm), Warrenton (166.9 mS/m; 867 ppm) and Ganspan (83.3 mS/m; 421 ppm) boreholes. Warrenton borehole had a high sulphate level (450 mg/l) as well. High chemical oxygen demand was observed in the Blaauwbank (62 mg/l) and Warrenton (98.5 mg/l) boreholes. In the dry and cold season similar observations were made for the various boreholes. Electrical conductivity and salinity levels remained high for the Trimpark (70.1 mS/m; 427.5 ppm), School (127 mS/m; 645 ppm), Warrenton (173.3 mS/m; 896.5 ppm) and Ganspan (88.1 mS/m; 444.5 ppm) boreholes. Nitrate levels for the Trimpark (14.1 mg/l) and School (137 mg/l), as well as sulphate levels for the Warrenton (325 mg/l) borehole were also high. Total coliforms, faecal streptococci and HPC bacteria were enumerated from water samples from all boreholes, except Blaauwbank where no faecal streptococci were enumerated. Faecal coliforms were enumerated from 5 of the possible 7 boreholes during a warm and wet season (Trimpark – 42 cfu/100ml; School – 2 cfu/100ml; Cemetery – 175 cfu/100ml; Warrenton – 3.84 x 10³ cfu/100ml; Ganspan – 1.9 x 10³ cfu/100ml). Indicator bacteria (FC, TC, HPC) exceeded target water quality ranges (TWQR) for drinking water in

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ii each case. During the cold and dry sampling season, faecal coliforms were enumerated mainly from the Trimpark (11 cfu/100ml) borehole. Total coliforms, faecal streptococci and HPC bacteria were enumerated from all the boreholes, except for Blaauwbank that contained no faecal streptococci or total coliforms. Enumerated indicator bacteria levels again exceeded TWQR for domestic use. Total coliform counts for the Pad dam borehole, however, complied with TWQR for domestic use. Identified E. coli were resistant to Erythromycin, Cephalothin and Amoxicillin and susceptible to Ciprofloxacin. Escherichia coli isolated from the Mooi River catchment shared the same antibiotic resistance phenotype. The most abundant HPC bacterial genus identified was Pseudomonas spp. (7 isolates). Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying degrees of antibiotic resistance were observed. Generally, the same pattern between the same genera were observed. All HPC isolates were resistant to Cephalothin and Amoxicillin and a lower degree Erythromycin and Streptomycin. The most abundant amoeba resistant bacteria was identified as Pseudomonas spp. Other isolates included Alcaligenes faecalis and Ochrobactrum sp. and Achromobacter sp.. All of these are opportunistic pathogens, except for Achromobacter. Resistance to more antibiotics (Streptomycin, Chloramphenicol, Cephalothin, and Amoxicillin) was observed in ARBs compared to HPC (Cephalothin, Amoxicillin) from bulk water from the same borehole. The water of all the aquifers sampled is of very poor physico-chemical or microbiological quality or both. Water may be used for irrigation or livestock watering only in the case where these boreholes comply with TWQR for said purposes. Results obtained indicate that the groundwater is faecally contaminated. Amongst the bacteria, opportunistic pathogens displaying various degrees of antibiotic resistance were frequently isolated. These results indicate health risks if untreated groundwater is consumed. Therefore groundwater needs to be treated before distribution especially if the water is for human consumption.

Keywords: Groundwater, rural communities, microbiological water quality, HPC, amoeba resistant bacteria, antibiotic resistance, opportunistic pathogens, 16S rRNA gene sequencing, mdh, lacZ.

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iii

Graag dra ek hierdie werkstuk op aan my Ma en Pa vir al die liefde en

ondersteuning, asook my Vader in die Hemel, sonder wie se bystand hierdie

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ACKNOWLEDGEMENTS

I would like to express my sincere appreciation to the following people and institutions for their contribution towards the completion of this project:

Prof. Carlos Bezuidenhout for all the guidance and advice given during the project; Water Research Commission for the funding of the project;

NWU for the bursaries and laboratory facilities;

All of the personnel of the Microbiology department for each of their individual contributions;

Karen Jordaan and Hermoine Venter for assistance given during sequencing of isolates: Guzene O‟Reiley and Herman Myburgh for assistance during the sampling sessions; My parents for financial support and patience;

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DECLARATION

I declare that this dissertation for the degree of Master of Science in Environmental Science (M.Sc Env.Sci) at the North West University: Potchefstroom Campus hereby submitted, has not been submitted by me for a degree at this or another university, that it is my own work in design and execution, and that all material contained herein has been duly acknowledged.

______________________ ________________

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LIST OF FIGURES

Figure 1.1: Average rainfall patterns of the North West province.

p 4 Figure 1.2: Pie chart displaying percentage of water used by the different

sectors in the province. p 7

Figure 2.1: Google map displaying the position of the three boreholes sampled

in the Potchefstroom municipal district. p 30

Figure 2.2: Google map displaying the position of the borehole which is

situated near the Klerkskraal dam. p 30

Figure 2.3: Google map displaying the location of the borehole situated on the

Blaauwbank farm. p 31

Figure 2.4: Google map of the two boreholes situated in the Harts River

catchment area. p 32

Figure 2.5: Photo illustrating all the instruments used during sampling in the

rear of the bakkie. p 33

Figure 3.1: A 1.5% (w/v) agarose gel illustrating the results of the multiplex

PCR. p 52

Figure 3.2: RDA ordination triplot of environmental parameters (red vectors) and biological data (blue vectors) for the various boreholes sampled.

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LIST OF TABLES

Table 3.1: Physico-chemical results for the Mooi River catchment area during a warm and wet season

p 41

Table 3.2: Physico-chemical results for the Mooi River catchment area during a cold and dry season

p 43

Table 3.3: Physico-chemical results for the Harts River catchment area during a warm and wet season

p 44

Table 3.4: Physico-chemical results for the Harts River catchment area during a cold and dry season

p 45

Table 3.5: Microbiological results for boreholes sampled in the Mooi River catchment area during a warm and wet season

p 46

Table 3.6: Microbiological results for boreholes sampled in the Mooi River catchment area during a cold and dry season

p 47

Table 3.7: Microbiological results for boreholes sampled in the Harts River catchment area during a warm and wet season

p 48

Table 3.8: Microbiological results for boreholes sampled in the Harts River catchment area during a cold and dry season

p 49

Table 3.9: Antibiotic resistance results of isolated E. coli from boreholes sampled in the warm and wet season

p 50

Table 3.10: Antibiotic resistance results of isolated E. coli from boreholes sampled in the cold and dry season

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xi Table 3.11: Identification and antibiotic resistance results of HPC bacteria isolated

from the Pad dam borehole

p 55

Table 3.12: Identification and antibiotic resistance results of HPC bacteria isolated from the Blaauwbank borehole

p 56

Table 3.13: Identification and antibiotic resistance results of HPC bacteria isolated from the Trimpark borehole

p 57

Table 3.14: Identification and antibiotic resistance results of HPC bacteria isolated from the Cemetery borehole

p 58

Table 3.15: Identification and antibiotic resistance results of HPC bacteria isolated from the School borehole

p 59

Table 3.16: Identification and antibiotic resistance results of HPC bacteria isolated from the Warrenton borehole

p 61

Table 3.17: Identification and antibiotic resistance results of HPC bacteria isolated from the Ganspan borehole

p 62

Table 3.18: Identification and antibiotic resistance results of amoeba resistant bacteria isolated from the Pad dam borehole

p 64

Table 3.19: Identification and antibiotic resistance results of amoeba resistant bacteria isolated from the Cemetery borehole

p 65

Table 3.20: Identification and antibiotic resistance results of amoeba resistant bacteria isolated from the School (SK) borehole

p 66

Table 3.21: Identification and antibiotic resistance results of amoeba resistant bacteria isolated from the School borehole

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1

CHAPTER 1 – GENERAL INTRODUCTION AND LITERATURE

REVIEW

1.1 INTRODUCTION

South Africa is situated in a semi arid region of the world. The average rainfall of the country (450 mm) is far below the average rainfall per year of the world (NWRS, 2004). In relation to the country as a whole, the North West province has very little perennial surface water resources, and the little that is available is under a large threat of pollution due to anthropogenic activities (NWPG, 2002; DWAF, 2006).

People living in rural areas are largely dependent on groundwater resources to meet the demand for water used for domestic purposes. In the North West province, this amounts to 65% of 3.5 million people (DWA, 2009). As groundwater was previously considered to be of excellent quality (physico-chemically and microbiologically), this resource was usually supplied to communities without prior treatment (Momba et al., 2006). Recent studies in the North West province (Ferreira, 2011) and Gauteng (Mwabi et al., 2012) concluded that this perception of groundwater quality is incorrect. A high percentage of boreholes in the North West province that was tested (Ferreira, 2011) did not comply with target water quality ranges (TWQR; physico-chemical and microbiological) for drinking water (DWAF, 1996a) and none of the water tested by Mwabi et al. (2012) complied with the same microbiological TWQR. These examples demonstrate that the monitoring of groundwater resources needs to be performed to prevent health implications.

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2 1.2 RESEARCH AIM AND OBJECTIVES

The aim of this study was to evaluate the microbiological and physico-chemical quality of water from aquifers in the North West province, South Africa.

The objectives of the study were to:

i. Collect water samples from DWA monitoring boreholes

ii. Analyse water samples on site for set physico-chemical parameters using appropriate instruments

iii. Analyse water samples for levels of faecal indicator organisms iv. Isolate and identify heterotrophic plate count bacteria

v. Identify amoeba resistant bacteria

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3 1.3 LITERATURE REVIEW

1.3.1 Water and groundwater availability

Water is less available on the African content than in Asia, Europe or North America and with one of the fastest urbanisation rates in the world, this could lead to a major water shortage (Showers, 2002).

South Africa is situated in a semi-arid region of the world (NWRS, 2004). Climatic conditions associated with semi-arid regions include low average precipitation, where South Africa has an average of 450 mm/year compared to the 860 mm/year of the world (NWRS, 2004). High evaporation rates, high temperature, large variability in precipitation and low mean annual runoff contribute to stresses already placed on water resources (NWPG, 2002; NWRS, 2004).

South Africa is mainly dependent on surface water sources for irrigation, industrial and urban water supply (NWRS, 2004). For this reason, the surface water infrastructure is well developed (NWRS, 2004). The reliable local yield for the year 2000 was estimated at 13227 million m³/annum and local requirements at 12 871 million m³/annum, which gives a surplus of 186 million m³/annum (NWRS, 2004). From estimations (under a scenario of high population and economic growth) for local water requirements in the year 2025, a water deficit of 2044 million m³/annum is predicted (NWRS, 2004). Parsons (2003) estimated that ground water can supply a reliable yield of 19 250 million m³/year, from which only 2 100 million m³/annum is abstracted.

The North West province has a surface area of 116 320 km² with a geology of igneous, ancient igneous volcanic and sedimentary rocks (NWPG, 2002). From Figure 1.1, it can be observed that the rainfall averages are highly variable and decrease from the East (600 mm) to the West (less than 300 mm) (NWPG, 2002). Climatic conditions are characterised by high evaporation rates that exceed precipitation rates and a low average mean annual run-off (6% compared to 9% of South Africa) (NWPG, 2002). Perennial water sources

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4 (Crocodile, Marico and Vaal Rivers) are concentrated to the South Eastern and North Eastern parts of the province, where these water sources are shared with bordering provinces (NWPG, 2002).

Figure 1.1: Average rainfall patterns of the North West province (DWA, 2010)

Surface water sources for the province include rivers (Crocodile, Marico, Vaal and tributaries [34]), dams, pans, wetlands and dolomite eyes (NWPG, 2002). Although surface water resources are limited in the province, a large reservoir of groundwater is available (NWPG, 2002). These reservoirs are comprised of dolomitic compartments and fractured aquifers (NWPG, 2002). Average annual recharge of groundwater resources in the province is very low (less than 10 mm) and is the lowest in the country (NWPG, 2002). Water abstraction from aquifers in the province can range from as low as 0.1 – 1 million m³/ annum (Reivilo, Stella) to as high as 27 million m³/annum in the Louwna-Ganyesa region (NWPG, 2002).

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5 Because of the scarcity of surface water resources in the province, people are reliant on groundwater resources to fulfill the demand for water for domestic use. Sixty five percent of people from a population of 3465 million live in rural areas in the North West province, and rely solely on groundwater for domestic use (DWA, 2009; NWPG, 2002). This figure is in close correlation to the estimated 80% of the population in rural or low income communities in developing countries that rely on groundwater as sole source of drinking water (Murray et al., 2004). These figures indicate the importance of groundwater as a water resource.

Groundwater was classified as private water in the previous Water Act (Act no. 54 of 1956) of South Africa. Therefore there was no need to monitor the quality of groundwater. The new Water Act (Act no. 36 of 1998) identified groundwater as a national resource and therefore the need to monitor the water quality of groundwater resources. According to the Water Act (Act no 36, 1998) all water resource must be protected. Through monitoring of the quality of the groundwater, anthropogenic impacts on water resources can be identified and remediated, and so can the water resources be protected against deterioration through pollution.

Many sources of pollution exist in the province. These include agricultural practices, the mining industry, negative impacts of population growth and urbanisation (Coetzee et al., 2006; Griesel & Jagals, 2002). All of these sources could contribute to chemical and/or microbiological contamination and dewatering of aquifers (irrigation, mining) (NWPG, 2002; Coetzee et al., 2006).

Groundwater is perceived as being inherently of pristine quality (Mpenyana-Monyatsi and Momba, 2012; Momba et al., 2006; Ferguson et al., 2012; Mackintosh & Colvin, 2003). For this reason, many of the rural areas in South Africa are supplied with groundwater for domestic use without prior treatment (Lehloesa & Muyima, 2000; Mackintosh & Colvin, 2003). This scenario would place people using the untreated groundwater at a high risk of exposure to chemical contaminants and pathogens which would lead to adverse health effects.

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6 1.3.2 Water dependent economic activities in North West province

Because of rich ore deposits in South Africa, mining has become an important economic activity (Coetzee et al., 2006). The mining sector is the largest contributor (35.5%) to the economy as well as the largest employer (22%) (NWPG, 2002). Mining plays an important part in hydraulic environments, whether active or inactive, where both states could negatively affect water resources in several different ways (DWAF, 2003b). The mining industry consumes large amounts of water for process purposes and discards polluted used water into streams, rivers or tailings dams from where toxic metals leach to groundwater resources (Winde, 2010). Mining activities cannot be safely performed under dolomite compartments, due to their large water storage capacity. Therefore the compartments (aquifers) are dewatered, leading to loss of local available groundwater yield (NWRS, 2004).

The West and East Rand basins in Gauteng contain highly polluted groundwater, due to mining activities. During dewatering, these waters are decanted into surface water resources that may infiltrate groundwater resources (Winde, 2010). It is a well known fact that ground and surface water interacts with one another (Xu et al., 2002; Parsons, 2003; Le Maitre & Colvin, 2008). Contamination of one source would inevitably lead to the contamination of the other due to physical interactions. These decanting mine waters contain high concentrations of total dissolved salts (TDS), sulfates (acid mine drainage), sodium, chloride and nitrates (DWAF, 2006). Thus, return flow water from mines is usually of poor quality, which affects downstream users using the water for domestic purposes (Coetzee et al., 2006). Therefore, it is imperative that mine water effluents and return flow water be of good quality.

Agriculture is recognised as the biggest consumer of groundwater resources (Fig. 1.2) for irrigation use, as well as the occupier of the largest surface area of the province (livestock farming) (Conrad et al., 1999; NWPG, 2002). Although being the largest consumer of water, the agricultural sector only contributes a small percentage to the economy of the province. This is indicative of poor water use efficiency (NWRS, 2004). Over-abstraction of groundwater can lead to the depletion of localised groundwater resources and irrigation

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7 return flows may contain a wide variety of pesticides, herbicides and leachable ions due to incorrect fertiliser applications (Harold & Bailey, 1996). Livestock farming (feedlot runoff) can contribute to antimicrobial and faecal pollution of groundwater resources (Alanis, 2006).

Urbanisation and population growth also exert pressure on available water resources (NWRS, 2004). A high influx of people to urban areas results in informal settlements expanding where sanitary infrastructure is usually lacking (DWA, 2003). On site sanitation is normally incorporated for human waste disposal in these circumstances (Mpenyana -Monyatsi & Momba, 2012). On site sanitation has been identified as a contributor to faecal pollution of groundwater resources (Godfrey et al., 2005; Bonton et al., 2010; Howard et al., 2003).

Fig 1.2: Pie chart displaying percentage of water used by the different sectors in South Africa (Data obtained

from NWRS, 2004).

Where sewerage is available, many of the waste water treatment plants of the province do not adequately treat the water to remove microbes from the water. Twenty of the 32 waste water treatment plants in the province do not comply with 50% of the TWQR for microbiological removal set by the Department of Water affairs (DWA, 2012). Water

Irrigation Urban Mining Rural Afforestantion Power gen.

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8 containing faecal bacteria and possibly pathogens are released into receiving water bodies exposing people downstream to these pathogens.

1.3.3 Factors influencing groundwater recharge (contaminant movement)

Topography of soils plays an important part in the absorption of water into soil. Flat surfaces would allow more time for precipitation to penetrate soil. More precipitation absorbed would allow more contaminants to be transported to groundwater sources, for example, nitrates. In contrast, steep soils would allow less precipitation to penetrate soil (Iqbal & Krothe, 1995).

The geological composition of the unsaturated and saturated zones above underground water resources also determines the quality of water entering the aquifer. Rock formations made up of coarser grains have a higher permeability and porosity as opposed to rock formations with a finer/fine grained composition (Yasmin, 2009). Coarse grained rocks would therefore allow fast and easy movement of water through the zones and less attenuation of contaminants and vice versa for finer grained rock formations (DWAF, 2003a). The physical properties of a specific aquifer therefore play an important role in the vulnerability to, and the rate and time of contamination (Valenzuela et al., 2009; Krapac et al., 2002; Gelinas et al., 1996).

Borehole construction should also be considered when contamination of groundwater resources is investigated. Poor borehole construction would lead to easier groundwater contamination, as all of the inherent attenuation processes described above are bypassed. After installation of the casing of a borehole, a gravel pack needs to be installed between the actual wall of the hole in the ground and the casing. Materials used for this purpose should be of an inert nature as to not change the water quality entering the borehole. Quartzitic gravel is the most commonly used material (Barnes & Vermeulen, 2012). A sanitary seal then needs to be fitted from the ground level down to 5 meters (Barnes & Vermeulen, 2012). This is to prevent surface runoff water from entering a groundwater resource through the space between the casing and the hole in the ground (DWAF, 2003a). Bentonite is the material of choice as it forms an impermeable layer (Barnes & Vermeulen,

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9 2012). Lastly, a concrete slab needs to be placed around the protruding casing in such a manner that water will not pool around the casing (DWAF, 2003a; Barnes & Vermeulen, 2012).

1.3.4 Surface and groundwater interactions

Considering that there are interactions between ground and surface waters, surface water can have a significant impact on groundwater (DWAF, 2003b; Xu et al., 2002). This interaction is dependent on the relative level of the water referred to. For example, surface water would recharge ground water when the level of the surface water is higher than that of the groundwater (Gardner, 1999; Winter et al., 1998). This interaction usually occurs at an upper catchment level (near origin of river, stream) (Xu et al., 2002). The opposite applies in a lower part of the catchment, where groundwater may contribute to river flow (Parsons, 2003). These are examples of change over time in the system (Gardner, 1999).

During precipitation, and subsequent recharge of groundwater, the water level of groundwater can rise above that of a relevant surface water body. Groundwater will then move to the surface water body (Le Maitre & Colvin, 2008). This would be an example of change over time (Gardner, 1999). Ground and surface water interactions are not limited to rivers alone. Groundwater sources can contribute water to rivers in the form of springs and interact with wetlands (Parsons, 2003). The interactions of surface and groundwater sources are controlled by the geology between the separate water bodies (Le Maitre & Colvin, 2008). High transmissivity and porosity values (fast movement and large storage) would be indicative of a more pronounced interaction between water bodies (Le Maitre & Colvin, 2008). All surface and groundwater bodies are however, connected to some degree (Parsons, 2003). Because of these interactions, contamination or pollution of surface water can contribute to groundwater contamination and vice versa (Gardner, 1999; Winter, et al., 1998).

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10 1.3.5 Types of pollution

There are mainly two types of water pollution that should be considered, namely microbiological and chemical pollution. The former includes pollution by viruses, bacteria, protozoa and helminths. Chemical pollutants are carbon, nitrogen, phosphorous and various other minerals and metals (Fourie & van Ryneveld, 1995).

1.3.5.1. Chemical pollution.

1.3.5.1.1 pH.

Rivers and other water sources are buffered by natural buffering systems, so as to allow that the pH stays in close ranges to a neutral pH of 7 (Dallas & Day, 2004). This happens because of the action of complex acid-base equilibria of various dissolved compounds, mainly the carbon dioxide-bicarbonate-carbonate equilibrium system (DWAF, 1996a). The pH of water thus does not indicate the ability of the water to neutralise additions of bases or acids. When acids or bases are added to natural water, some elemental composition changes in water take place. For example, aluminium occurs as unavailable hydrated hydroxides in alkaline waters, and as the pH drops, it is converted to the highly toxic Al3+ ion (Dallas & Day, 2004). Changes in the pH of water also cause some elements to be more or less available, through altering their solubility. A decrease in pH will bring about a surface charge change of an ion or molecule, making the ion or molecule more soluble in water. This may then lead to the release of toxic substances from the sediments of water bodies (Dallas & Day, 2004). Sources of pollution that could change the pH of water include acid mine drainage and industrial processes (DWAF, 2006).

The TWQR for drinking water established by DWAF (1996a) are a pH range of between 6.0 to 9.0. Deviation from this range to more acidic levels would bring about severe danger due to toxic metal ions while deviation to more alkaline levels poses danger due to deprotonated species (DWAF, 1996a). The TWQR for irrigation purposes are between 6.5 and 8.4(DWAF, 1996c). Using water not complying with these TWQR will result in foliar damage of crops (DWAF, 1996c).

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11 1.3.5.1.2 Total Dissolved Solids.

Total dissolved solids (TDS) in water originate from enhanced weathering of minerals from acids in the soil, process water effluent and fissure water effluent (Atekwana et al., 2004; Coetzee et al., 2006). Atekwana and colleagues (2004) concluded that TDS is a likely geochemical parameter that closely links bulk electrical conductivity to microbial degradation of hydrocarbons. Electrical conductivity is directly proportional to the concentration of TDS in a water sample by a factor of 6.5 (Atekwana et al., 2004; DWAF, 1996a). TDS can be measured by automated meters or the gravimetrical method. In the latter case, the water sample is evaporated and the solids measured that are left behind (Atekwana et al., 2004).

Various methodologies are available to remove bulk TDS from contaminated water. These are usually based on membrane separation processes and include nano filtration, reverse osmosis and electro-dialysis with bi-polar membranes (Basha et al., 2008; Chandramowleeswara & Palanivelu, 2006). The TDS in water does not cause any adverse human health effects. It however affects the aesthetic value of the water (DWAF, 1996a).

Target water quality ranges for domestic water is set at less than 70 mS/m for electrical conductivity (DWAF, 1996a). Higher concentrations will lead to a disturbance in the salt balance of the body (DWAF, 1996a). Livestock can tolerate water with a TDS concentration of up to 4000 mg/l (DWAF, 1996b). Elevated levels would lead to a decrease in production, as animals would be reluctant to drink such water (DWAF, 1996b). Crops can be irrigated with water having an electrical conductivity of up to 40 mS/m, where elevated levels would lead to decreased productions in sensitive crops (DWAF, 1996c).

1.3.5.1.3 Nitrates.

Nitrates may contaminate groundwater, as well as surface water sources. This could be due to the result of incorrectly treated waste water disposal, industrial practices and agricultural practices, where too much nitrogen fertilisers are added to the soil for efficient uptake by plants (Smith et al., 2005; Moreno et al., 1996; Suthar et al., 2009; Shroder et al., 2004).

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12 Nitrate is water soluble and therefore readily transported through sediments into groundwater (Aelion et al., 1997; Tesoriero et al., 2007; Andrade & Stigter, 2009). Other sources of nitrogen pollution include livestock farming (animal waste), chemical industrial effluents high in nitrogen, pit latrines and landfills (Suthar et al., 2009; Al-Khatib & Arafat, 2009; Wakida & Lerner, 2005).

Accompanied with the varying types of rainfall associated with semi- and arid regions, nitrogen in the soil will leach out much faster during times of heavy precipitation when water flow through the soil is fast, causing increased nitrogen levels in groundwater resources (Tredoux, 2004; Stigter et al., 2008). Other factors that also affect the movement of nitrogen include the transmissivity of soils and aquifers (Stigter et al., 2008).

Problems associated with high nitrate concentrations in water (+20 mg/ml) include methaemoglobinanemia, cancer and headaches (Tredoux, 2004; Almasri & Kaluarachi, 2004; Suthar et al., 2009; Wakida & Lerner, 2005; Faniran et al., 2001). When infants ingest large quantities of water containing high concentrations of nitrates, the nitrates would first be converted to nitrite by microbes in their digestive systems (Suthar et al., 2009). The nitrites are absorbed into the blood stream where they bind to haemoglobin to form methaemoglobin. Infants do not have the metabolic pathways to detoxify the methaemoglobin (Wright et al., 1999). Methemoglobin decreases the oxygen carrying capacity of blood and infants may, in the worst case scenario, die because of asphyxiation (Tredoux, 2004; Suthar et al., 2009).

Cancers of the digestive tract may also be caused by nitrates. Nitrites that are formed endogenously in the digestive tract of humans undergo nitrosation reactions in the stomach with amines to form a variety of N-nitroso compounds which are carcinogens (Suthar et al., 2009; Tredoux, 2004). This is, however, a controversial issue and further research and evidence is required. Nitrates also acts as vasodilators of the cardiovascular system. When nitrates are ingested through water or any other medium, the arteries in the body would dilate and a headache would develop (Suthar et al., 2009; Tredoux, 2004).

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13 Cattle are also at risk when ingesting water with high nitrate levels. Although cattle can tolerate higher nitrate concentrations than humans, negative health effects are also evident in constant exposure to high concentrations. These include increased indices of abortion, infertility and inhibition of growth. Death of cattle could also occur when there is a sudden extreme spike in the nitrate concentration of water (Tredoux, 2004; Suthar et al., 2009).

Water used for domestic purposes should contain less than 10 mg/l of nitrates (DWAF, 1996a). Elevated levels would increase the occurrence of methemoglobinanemia. Sensitive crops can tolerate water containing up to 5 mg/l of nitrates and most other crops a nitrate concentration of up to 30 mg/l (DWAF, 1996b). Higher levels would negatively affect yields. The TWQR for livestock water is set at 100 mg/l (DWAF, 1996c). Water containing levels of between 200 and 400 mg/l would affect monogastrics and ruminant animals alike (DWAF, 1996c).

1.3.5.1.4 Sulphate

In South Africa, gold bearing ore contains about 3% pyrite (FeS2), which was and is still being transported to the surface due to mining activities (Tutu et al., 2008). To extract gold from ore, a cyanidation process is used, because it is very selective to gold, leaving behind all other minerals contained in ore (Naicker et al., 2003). Unused slurry of this process is pumped to tailings dams where it is exposed to the environment and oxygenated rain during precipitation events (Coetzee et al., 2006). Pyrite and other sulphur containing minerals are oxidized in the tailings dams and sand heaps (artifact of gold extraction method used prior to cyanidation process) (Tutu et al., 2008). Acidic water percolates to groundwater sources which feed streams in lower lying areas surrounding affected areas (Naicker et al., 2003). Pyrite is also exposed to oxygen in abandoned mines

Sulphate is a normally occurring compound found in geologic formations and therefore in water as well with fluctuating concentrations (Naicker et al., 2003). Mining activities expose pyrite to oxygen during which pyrite is oxidized into sulphate and an acid (Tutu et al., 2008). Acid and sulphate containing water percolates to groundwater (tailings dams and sand heaps) or decants into surface water sources contaminating such resources (Coetzee et al., 2006). Such water may contain sulphate levels of up 7500 mg/l (Tutu et al.,

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14 2008). Sulphates are also contributed to surface water sources by acid rain produced by anthropogenic activities (DWAF, 1996a).

Initial exposure to water containing high levels of sulphates will cause diarrhea, but individuals are able to adapt to consuming water with elevated levels of sulphate (DWAF, 1996a). No adaptation will, however, occur when water with sulphate levels of 1000 mg/l and higher are consumed (DWAF, 1996a).

1.3.5.2 Microbial pollution.

Bacteria occur naturally in groundwater and most of these are not pathogenic to humans or animals. Only when pathogens are present in water sources will disease outbreaks occur. Conditions presiding in groundwater favor bacterial growth and survival and could allow pathogenic organisms to survive for long periods (Murray et al., 2004).

Sources of bacterial pollution include pit-latrines, incorrect sewage discharges and spills, landfill leachate and agriculture practices such as intensive cattle/sheep/dairy farming. It could also result from recharge (natural or induced) of aquifers by polluted water sources (Crowther et al., 2002; Howard et al., 2003; Murray et al., 2004; Field & Samadpour, 2007).

Factors that influence the movement of micro-organisms through soil are grain size, soil composition (clay, loam or sandy soil), mineral content of soil, residence time of water in soil and the chemical conditions presiding in soil (Flynn & Sinreich, 2010; Fourie & van Ryneveld, 1995).

Bacteria are usually filtered out of water by soil, and thus, the further away a water resource from a contaminant source, the less chance of contamination exist (DWAF, 2003a). Soil with small grain sizes (clayey soils) will remove (attenuate) micro-organisms more effectively than soils with larger grain sizes (sandy soil) as the retention time of water in the clayey soils is longer (DWAF, 2003a).

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15 Larger micro-organisms, such as helminths and protozoa, would be efficiently filtered out of water if the soil has a loam or clayey-loam composition (Fourie & van Ryneveld, 1995). Smaller micro-organisms, such as bacteria and viruses, would only effectively be filtered out of water by clay/clayey soils. Micro-organisms mobility through soil is inversely proportional to their physical size. The time that it takes the micro-organisms to reach groundwater sources also plays an important role. Micro-organisms can only survive in soil for a finite period of time, and if the travel times exceed between 150 – 200 days, contamination from micro-organisms (bacteria) should not pose a health threat (DWAF, 2003b).

Strong correlations were observed between short term rainfall before sampling and the amount of indicator bacteria found in groundwater (Howard et al., 2003; Fourie & van Ryneveld, 1995). Micro-organisms get trapped to the soil particles due to the difference in surface charges of the soil particles and the bacteria (Murray et al., 2004; Howard et al., 2003). The bacteria can, and will be, flushed out of soil during heavy precipitation events, where the micro-organisms will then end up in groundwater sources as aquifers are recharged during or after precipitation (Howard et al., 2003; Fourie & van Ryneveld, 1995). Permeability of soils to bacteria also increase as the soil becomes more saturated with water, causing bacterial cells to travel along the saturated soil into groundwater resources (Howard et al., 2003).

In soils, there are artificial channels that are made by decomposed roots and organisms such as earthworms. In rock formations fissures are present, and these channels present water with a pathway by which it can flow freely and where all the processes of attenuation are bypassed. This type of water movement is termed as macropore flow (Fourie & van Ryneveld, 1995). It would allow for the contamination of groundwater resources, irrespective of the depth of soil between a contamination source and groundwater resource (Fourie & van Ryneveld, 1995).

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16 1.3.6 Indicator organisms

Most human pathogens are transported via the faecal oral route (WHO, 2011). Pathogens are then excreted in faeces from infected people. When faecal matter is not disposed of correctly, pathogens may be transported to water sources (surface or ground). When the correct physico-chemical environment presents itself, pathogens can then survive in water. Ingestion of contaminated water may lead to infection by these pathogens to cause adverse health effects, such as cholera, typhoid fever, paratyphoid fever, bacillary dysentery and diarrheal disease (Lawrence et al., 2001; Fourie & van Ryneveld, 1994).

A wide range of pathogens could be present in water after contamination, and to test water for all known pathogens would not be economically viable or productive (Baghel et al., 2005). Indicator organisms have therefore been identified to indicate possible faecal contamination of water sources.

Micro-organisms have to comply with certain criteria before being incorporated as an indicator organism. No one micro-organism complies with all of the set criteria, and therefore a selection of specific indicator organisms are used in conjunction with one another to obtain reliable results on the microbiological state of waters. Commonly used indicators occur together with enteric micro-organisms in the intestines of warm blooded animals and are therefore excreted together with enteric pathogens. Indicator organisms can then be used to analyse water for faecal pollution (Willey et al., 2011; Atlas & Bartha, 2002; DWAF, 1996a). Micro-organisms considered to be used for indicator organisms should comply with the following criteria i) Should be present in faeces of warm blooded animals ii) Should be easily detectable using simple methods iii) Should not multiply in water sources or in any other environmental settings iv) Should be present in water together with pathogenic organisms v) Should have the same or longer die-off rate than pathogenic organisms and should not cause adverse health effects in humans (DWAF, 1996a; NHMRC, 2003; Atlas & Bartha, 2002). Using this criteria there are four generally used indicator organisms, namely total coliforms, faecal coliforms, faecal streptococci and heterotrophic plate count bacteria.

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17 (a) Total coliforms

These micro-organisms form part of the Enterobacteriaceae family and can be isolated from a variety of environments (NHMRC, 2003), including the gastro-intestinal tract of humans. Bacteria belonging to this group include the species Escherichia, Citrobacter, Enterobacter, Klebsiella and Serratia (DWAF, 1996a). All of these micro-organisms can multiply in the environment and water sources (Cohen & Shival, 1972; NHMRC, 2003) and are therefore not used exclusively for detection of faecal pollution in water sources (NHMRC, 2003). This group of indicator organisms is generally used to analyse the general sanitary quality of water, as well as to indicate possible failures in distribution systems (DWAF, 1996a; NHMRC, 2003) and may indicate the potential presence of entero pathogens in water (Rompre et al., 2002). The TWQR for these indicator organisms are less than 5 cfu/100 ml for domestic use (DWAF, 1996a). Levels higher than 100 cfu/100ml would lead to a significant increase in risk of infectious disease transmission (DWAF, 1996a).

(b)Faecal coliforms

Faecal coliforms are part of a subgroup of total coliforms. These bacteria are distinguished from the total coliform group in that they can grow at elevated temperatures of 44.5°C (Paruch & Maehlum, 2012; Rompre et al., 2002). Incubation at this temperature inhibits growth of non faecal coliform bacteria (Rompre et al., 2002). Escherichia coli are part of this group of indicator organisms and are the only faecal coliforms that are exclusively present in the intestinal tract of warm blooded animals (Foppen & Schijven, 2006; Paruch & Maehlum, 2012). Escherichia coli also outnumber the other faecal coliforms of the gastro-intestinal tract and comply with most of the criteria set for selecting an indicator organism (Foppen & Schijven, 2006). Gabutti et al. (2000) stated that E. coli detection in water samples would only indicate recent faecal pollution, as faecal coliforms (E. coli) are not very persistent in environmental conditions. For these reasons E. coli is recognised as the best indicator of faecal pollution in water sources (Paruch & Maehlum, 2012).

Target water quality ranges for faecal coliforms are set at zero for domestic use, less than 200 cfu/100ml for livestock watering purposes and up to one cfu/100ml for water used for irrigation (DWAF, 1996a; DWAF, 1996b; DWAF, 1996c). Increased levels of up to 10

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18 cfu/100ml would have an increased risk of infectious disease transmission if water is used continuously by humans. Levels of more than 200 cfu/100ml would increase the chances of infectious disease transmission in animals. Crops irrigated with water containing up to 1000 cfu/100ml would cause transmission of infectious disease if crops like vegetables are consumed raw. Water with this level of faecal contamination can, however, be used to irrigate parks and tree plantations if human contact with this water is avoided (DWAF, 1996a; DWAF, 1996b; DWAF, 1996c).

(c) Faecal streptococci

These micro-organisms include four species of the genus Enterococci. They include Enterococcus faecalis, E. faecium, E. durans and E. hirae (Junco et al., 2001). These indicator organisms are also present in the faeces of warm blooded animals and are more persistent in the environment than E. coli (Gabutti et al., 2000; Willey et al., 2011). Geldreich (1996) stated that faecal streptococci are more abundant in faeces from warm blooded animals than faecal coliforms. Faecal streptococci are also more persistent in environmental conditions (Gabutti et al., 2000). Faecal streptococci would therefore give an indication of faecal pollution that occurred a longer time ago if no faecal coliforms are enumerated together with faecal streptococci. No TWQR are set for this indicator organism.

(d) Heterotrophic plate count bacteria (HPC)

This group of micro-organisms includes all naturally occurring bacteria that utilise organic nutrients at low concentrations for growth (Edberg et al., 1997; Edberg & Allen, 2004). These bacterial counts give an indication of the general microbial quality of water and do not indicate faecal pollution (DWAF, 1996a). Counts also do not give a representation of the total number of bacteria present in water (DWAF, 1996a). Heterotrophic plate count bacteria may also decrease the sensitivity of other methods to enumerate indicator organisms (Allen et al., 2004; Quiroz, 1999). Allen et al. (2004) stated that high numbers of HPC bacteria inhibit the growth of coliform and subsequent groups on selective media. This would obscure the presence of these important indicator organisms.

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19 Opportunistic pathogens do make out some of the heterotrophic bacteria and include the genus Pseudomonas, Acinetobacter, Flavobacterium, Alcaligenes, Achromobacter, Aeromonas and Mycobacterium (Quiroz, 1999; Payment et al., 1991; Stelma et al., 2004; Geldreich, 1996). These bacteria do not occur in enough numbers in water to cause gastrointestinal disease in healthy people, as no sufficient clinical evidence exists to suggest otherwise (Edberg et al., 1997; Edberg & Allen, 2004; Allen et al., 2004). Some researchers do not agree with this statement, as HPC bacteria with virulence factors were identified in water (Pavlov et al., 2004; Payment et al., 1991). Chances of developing disease due to ingestion of these opportunistic pathogens are higher in immune compromised individuals, the elderly and the very young (Ford, 1999; Paulse et al., 2009).

Heterotrophic plate count bacteria are used as an indicator of bacterial after growth or contamination that may have occurred after treatment of water (DWAF, 1996a). Higher than normal HPC counts may therefore indicate the possible presence of pathogenic micro-organisms in water distribution systems.

The absence of a faecal indicator organism does not necessarily prove that no faecal contamination occurred in water sources (Fourie & van Ryneveld, 1994), as high HPC concentrations may obscure the identification/enumeration of faecal indicator organisms, as stated above. In conclusion, it would be advisable that all of the indicator organisms should be used in conjunction with one another, as every indicator micro-organism only gives an answer to a piece of the puzzle. This would give a holistic view of the microbial quality of water. Domestically used water may contain levels of heterotrophic plate count bacteria of up to 100 cfu/100ml (DWAF, 1996a). Elevated levels would increase the possibility of infectious disease transmission if such water is consumed.

1.3.7 Amoeba resistant bacteria (ARB)

Amoebas are unicellular eukaryotes that are predators on bacteria in the environment (Moliner et al., 2010). Two developmental stages can be observed from amoeba, namely the trophozite stage and a cyst stage (Greub & Raoult, 2004; Thomas et al., 2008). The trophozite is the vegetative feeding form and the cyst is the resting form during

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20 unfavorable environmental conditions (Greub & Raoult, 2004). Amoebas feed on bacteria through phagocytosis, where bacterial cells are engulfed and digested by enzymes (Greub & Raoult, 2004).

The diversity and abundance of amoeba in water sources are dependent on certain environmental factors. These include temperature, moisture content in non water sources, pH and nutrient availability in the form of bacterial cells (Greub & Raoult, 2004). Previously identified ARB genera include Acinetobacter, Aeromonas, Bacillus Pseudomonas, Alcaligenes, Brevundimonas, Chryseobacterium, Comamonas, Delftia, Flavobacterium and Ochrobactrum (Pagnier et al., 2008; Barker and Brown, 1994).

Bacteria isolated from amoeba can be divided into three groups. Some are obligate intracellular bacteria, some are facultative intracellular and other are endosymbionts (Moliner et al., 2010; Thomas et al., 2008; Greub & Raoult, 2004). For bacteria to be able to survive in the phagolytic environment of amoeba, they have developed certain mechanisms of resistance. Specific mechanisms include the resistance to microbicidal effectors in the phagocytes of amoeba, the ability to replicate in the intracellular environment or the secretion of toxins that kill of the amoeba before phagocytosis can be completed (Thomas et al., 2006; Cosson & Soldati, 2008; Thomas et al., 2008; Greub & Raoult, 2004). The mechanism of phagocytosis used by amoebas is the same as macrophages incorporated by multicellular organisms (Greub & Raoult, 2004; Thomas et al., 2006; Cosson & Soldati, 2008). This would allow ARB to resist phagocytosis of the human body, which is one of the immunological responses to infection. Amoeba can also increase antimicrobial resistance of ARB (Pagnier et al., 2008), either by horizontal gene transfer (genes that encode for resistance) with other ARB, the amoeba host or free DNA of digested non resistant bacteria (Moliner et al., 2010). All of these mechanisms increase the pathogenicity of ARB. Amoeba resistant bacteria can therefore be considered as emerging human pathogens (Pagnier et al., 2008).

When bacteria are able to resist digestion by amoeba, the amoeba can become a reservoir of these bacteria (Thomas et al., 2006; Pagnier et al., 2008). As amoeba can resist harsh

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21 environments by forming a cyst, the amoeba would not be affected by disinfecting processes. These cysts forms of amoeba would then protect ARB against forms of disinfection that would normally kill bacteria. All of these factors would contribute to the spreading of inherently pathogenetic ARB bacteria.

1.3.8 Antibiotic resistance

Enteric pathogens are usually Gram negative rods (Alanis, 2006). Micro-organisms that have been routinely identified with antibiotic resistance include Shigella spp, Salmonella, Vibrio cholera, E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa (Alanis, 2006; Jansen et al., 2006). Bacteria possess the ability to adapt to their environment and therefore they can develop resistance to antimicrobials (Donskey, 2006; Alanis, 2006). Mechanisms of resistance include efflux pumps, expression of inactivation enzymes, modification of targets that antimicrobials bind to and making alterations to outer membrane proteins to inhibit antimicrobials from penetrating the intracellular environment (Donskey, 2006; Neu, 1992; Bax et al., 2000). Genes in the DNA of such micro-organisms encode for all of these resistance mechanisms.

When selective pressures (antimicrobial use) are applied by an environment, micro-organisms with mutations allowing them to survive unfavorable environments are selected for (Bax et al., 2000; Neu, 1992; Alanis, 2006). Micro-organisms with these mutations then transfer genes that encode for resistance to other micro-organisms (Donskey, 2006; Bax et al., 2000). The intestinal tract of humans provides a favorable environment for gene transfers and is also a reservoir for antibiotic resistant conferring genes (Donskey, 2006; Newman et al., 2011).

The misuse and over use of antibiotics leads to the emergence of antimicrobial resistant micro-organisms in the environment (Donskey, 2006; Alanis, 2006; Sundar et al., 2005). Antimicrobials are used clinically for humans and as growth promoters in the animal husbandry industry (Alanis, 2006; Sundar et al., 2005). It is possible to remediate this problem by correct and effective management strategies (Bax et al., 2000; Sundar et al., 2005).

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22 1.3.9 Principles of techniques available to study the quality of groundwater

1.3.9.1 Sampling collection

Water samples obtained from boreholes should be stored immediately on ice or at 4°C and analysed within six hours of sampling (Twarakavi & Kaluarachchi, 2006; Suthar et al., 2009; Griebler et al., 2010; Faniran et al., 2001; Howard et al., 2003; Crowther et al., 2002). Water should be pumped out of the borehole to ensure that a representative aquifer water sample is obtained and not stagnant water collected in the casing of the borehole (Bruce & McMahon, 1996; Suthar et al., 2009; Mclay et al., 2001).

1.3.9.2 Physico-chemical methods

1.3.9.2.1 Chemical parameters

Multiprobes can be used to measure the most physical parameters, including temperature, salinity, pH and electrical conductivity (EC) (Hydralab DS5). Dissolved oxygen concentration is also measured by using a handheld probe (sension156 probe, HACH, Germany). For the determination of chemical parameters, certain chemical reactions need to take place, which will be discussed accordingly.

(a) Nitrate

According to the HACH DR 2800 Spectrophotometer procedures manual (2007), nitrate concentrations in samples are measured using the cadmium reduction method. Cadmium reduces nitrate to nitrite. The nitrite then reacts with sulfanilic acid to form an intermediate diazonium salt in an acidic medium. The diazonium salt then reacts with gentisic acid to form an amber coloured solution. Absorbance of the sample is measured at 500 nm and results displayed in mg/l on the instruments screen.

(b) Sulphate

Suplhate in samples react with barium to form a barium sulphate precipitate. The resulting turbidity formed is directly proportional to the amount of sulphate present in samples. Absorbance is measured at 450 nm and results displayed as mg/l (HACH, 2007).

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23 (c) Chemical oxygen demand (COD)

Two milliliter aliquots of water samples are added to reaction vials supplied by the manufacturer. Vials are heated to 150°C for two hours. The reaction vials contain potassium dichromate, silver and mercury ions. The potassium dichromate acts as an oxidizing agent. Oxidisable organic compounds reduce the dichromate ion to a green chromic ion. The amount of Cr6+ left in the vials is measured at 420 nm and results displayed as mg/l on the instrument‟s screen (HACH, 2007).

1.3.9.3 Microbiological methods

1.3.9.3.1 Membrane filtration

Membrane filtration is defined by the US EPA (2005) as a vacuum or pressure driven separation process where particulate matter larger than 1 µm is rejected by using an engineered barrier. For detection of indicator organisms in water, sterile filters of pore size 0.45 µm is used (Cohen & Shuval, 1972; Wang & Wade, 2007). A water sample of 100 ml is filtered through the membranes and then aseptically placed onto selective media and incubated at the appropriate temperatures and time (Romprè et al., 2002). Nutrients and selective chemical compounds contained in the selective media diffuse through the filter to bacterial cells retained on the surface of the membrane (Wang & Wanda, 2007). Bacteria are counted and results given as colony forming units (cfu) per 100 ml.

1.3.9.3.2 Media

1.3.9.3.2.1 mFC Agar.

MFC agar is used for the detection and enumeration of faecal coliform micro-organisms by making use of the membrane filtration technique as described by Harley (2005). The agar plates are incubated at 45°C for 24 hours and the blue colonies on the agar plates are counted as faecal coliform bacteria (Grabow et al., 1981; Harley, 2005). The high incubation temperature makes the test more selective for faecal coliform bacteria (Finch et al., 1987).

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24 Yeast and peptone extracts serve as nutrient sources while bile salts inhibit growth of Gram positive bacteria (Merck, 2012). Rosolic acid contained in the agar serves as a pH indicator. Lactose fermentation by faecal coliforms produces acid to bring about colour change of pH indicator (Merck, 2012).

1.3.9.3.2.2 Membrane-lactose glucuronide agar (MLG agar).

MLG agar is used for the selective detection and enumeration of total coliforms and E. coli (Eccles et al., 2004; Fricker et al., 2008). The agar contains peptone and yeast extracts as nutrient sources and laurel sulphate to inhibit growth of Gram positive bacteria (Merck, 2012). Differentiation between coliforms and E. coli is based on two biochemical reactions occurring. Coliforms produce acid during lactose fermentation and turn the pH indicator phenol red to yellow (Merck, 2012). Escherichia coli produces the enzyme glucuronidase which cleaves 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide to from a blue chromofore inside cells (Merck, 2012). Coliforms are identified as yellow colonies and E. coli as green colonies on the agar (Eccles et al., 2004; Fricker et al., 2008; Harley, 2005).

1.3.9.3.2.3 KF-streptococcus agar.

KF-streptococcus agar is used for the selective isolation and enumeration of faecal streptococci (Po Cataloa Dionisia & Borrego, 1995; Domig et al., 2003). Feacal streptococci metabolise maltose and lactose to produce acid (Merck, 2012). During preparation of the agar, 1% of triphenyltetrazolium chloride is added to the agar, which is a stain that is assimilated by actively metabolizing cells, giving colonies a red to pink colour (Harley, 2005; Merck, 2012).

1.3.9.3.2.4 Mueller-Hinton agar.

This agar is used for antibiotic sensitivity testing to determine the susceptibility of bacteria to various antibiotics as well as the effectiveness of the different antibiotics to the bacteria. The test involves the growing of a bacterial matt on the medium on which paper disks are placed which are impregnated with an antibiotic. The antibiotic disks are standardised to contain a specific amount of antibiotic. The antibiotic will then diffuse into the agar and inhibit the growth of the bacterium (Leboffe & Pierce, 1999). Inhibition zones are then

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25 measured, recorded and compared to standards such as those from the National Committee for Clinical Laboratory Standards (NCCLS, 1999).

1.3.9.3.2.5 R2A agar.

R2A agar was used for the enumeration of heterotrophic bacteria contained in water (Reasoner & Geldreich, 1985). This medium has a low nutrient content to simulate environmental growing conditions and is incubated at lower temperatures (22°C - 25°C) for longer periods (5 – 7 days) (Merck, 2012). This is to allow for more accurate enumerations of stressed and chlorine tolerant bacteria and other heterotrophic organisms that develop slower (Merck, 2012).

1.3.9.3.3 Molecular based methods

The polymerase chain reaction (PCR) method is used to selectively amplify specific target genes of interest from small amounts of genetic material (Muyzer et al., 1993; Romprè et al., 2002; Willey et al., 2011).This is achieved by repeating three general steps cyclically. These include: i) Denaturing of DNA into single stranded DNA, ii) Annealing primers to single stranded DNA and, iii) Extension of the primers into new complementary DNA strands incorporating heat stable DNA polymerase and deoxyribonucleoside triphosphates (Romprè et al., 2002; Willey et al., 2011). These steps should be repeated for a minimum of 20 cycles up until enough product is produced. This molecular based technique is very rapid, specific and sensitive (Bej et al., 1990; Pollard et al., 1990; Romprè et al., 2002).

Due to very specific environmental conditions and micro-organisms adaptation to these conditions, not all micro-organisms can be grown on culture media. These uncultureable micro-organisms are termed as viable but noncultureable (VBNC) (Bej et al., 1990; Baggi et al., 2001). Owing to this fact, environmental samples may test negative for indicators although they are present in the samples in a VBNC state. The polymerase chain reaction procedure does not discriminate between the two states of micro-organisms and indicator or pathogenic micro-organisms will be detected none the less.

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26 Multiplex PCR refers to a PCR procedure where more than one gene target is identified in a single reaction (Romprè et al., 2002; Willey et al., 2011). The genes mdh and lacZ are housekeeping genes in E. coli (Bej et al., 1990; Romprè et al., 2002; Pupo et al., 1997; Reid et al., 2000). Multiplex PCR can therefore be used to screen for the presence of E. coli in samples, where both of these genes needs to be present for a sample to be positive for E. coli.

There are some drawbacks when using the PCR procedure. These include the fact that the procedure cannot determine that physiological state of organisms, as micro-organisms may be physiologically active or in a VBNC state. Environmental samples also contain substances that are inherently inhibitory to the PCR procedure, for example humic substances. Highly skilled staff is also required as well as specialised instruments and an equipped laboratory (Romprè et al., 2002).

The 16S ribosomal subunit is only found in prokaryotic organisms and archaea (Vandamme et al., 1996; Mignand & Flandrois, 2006). This subunit is also very conserved, with little variable regions occurring (Stackebrandt & Goebel, 1994; Lane et al., 1985). For these reasons this sub-unit has been used to identify bacteria from various sources via established databases (Mignard & Flandrois, 2006). Variations in DNA sequence results of the non conserved regions allow for the identification of bacteria and archaea (Vandamme et al., 1996).

Evaluation of microbiological and physico–chemical quality of water from aquifers in the North West Province, South Africa